Antibacterial and antifungal composition for agricultural and fisheries applications



United States Patent 3,247,062 ANTIBACTERIAL AND ANTHFUNGAL COMPOSI- TION FOR AGRICULTURAL AND FISHERIES 2 Claims. (a. 167-65) This invention relates to an antibacterial and antifungal composition for agricultural and fisheries applications which contains as the active ingredient a complex salt of kanamycin combined with a water-soluble inorganic metal salt.

The complex salt of kanamycin combined with a watersoluble metal salt according to the invention may be prepared as described hereinafter. The complex salt of kanamycin is sparingly soluble in water and shows not only an antibacterial activity owing to the kanamycin component but also an antifungal, anti-yeast activity; it is thus suitable for use in the agricultural application, including e.g. control of bacterial leaf blight of rice plant and in the fishing application, including e.g. control of diseases of trout caused by parasitic bacteria and fungi appearing in the culture of trout.

The term kanamycin used in the invention includes free kanamycin, salts of kanamycin such as kanamycin sulfate, hydrochloride and the like, as well as kanamycin B, salts of kanamycin B and mixtures containing one or more of the aforesaid substances. As the water-soluble metal salt there may be used many of water-soluble inorganic metal salts such as, for example, copper sulfate, nickel sulfate, cobalt chloride, zinc chloride, magnesium sulfate, ferrous chloride, etc. It is desirable that the watersoluble inorganic metal salt is contained in the complex salt in an amount of from about 0.5 to 3 mol. per mol. of the kanamycin component.

The complex salt of kanamycin and a water-soluble inorganic metal salt may be prepared by dissolving kana mycin or a material containing it in water, and subsequently adding to the solution a proper proportion of a water-soluble inorganic metal salt desired. The reaction may readily proceed at room temperature but it is also possible to promote the reaction by heating. The pH of the reaction medium may be then kept between about 4 and 8. Afterthe completion of the reaction, the solution may be evaporated to dryness but it is also possible to precipitate the complex salt of kanamycin by adding a Water-miscible organic solvent such as, for example, methanol, ethanol and the like.

The process of preparing the complex salt of kanamycin is illustrated below with reference to the following examples.

EXAMPLE 1 3.0 grams (0.005 mol.) of kanamycin sulfate 1a as 4 11 2 4 2 are dissolved in 25 cc. of distilled water by heating. 1.26 grams (0.005 mol.) of nickel sulfate (NiSO -7H O) are then added to the solution and uniformly dissolved therein. The resulting solution is allowed to leave overnight and then filtered. 120 cc. of ethanol are added to the filtrate to give the precipitate and 3.7 grams of the complex salt of the kanamycin sulfate with nickel sulfate are obtained. ";his complex salt obtained shows a potency of 617 meg/mg. and [a] =+95 (C. 0.5, H 0). shows the antibacterial and antifungal activities.

It further 3,247,062 Patented Apr. 19, 1966 EXAMPLE 2 On one hand, an aqueous solution of kanamycin sulfate is prepared as described in Example 1. On other hand, a solution of copper sulfate is prepared by dissolving 1.25 gr. (0.005 mol.) of copper sulfate (CuSO -SH O) in 3.5 cc. of distilled water. Both the solutions are combined together and the mixture becomes slowly blue-violet colored. After the mixture has been allowed to stand overnight, the solution is filtered and then precipitated by adding cc. of ethanol. The precipitate formed is removed out by filtration and 3.5 grams of the complex salt of the kanamycin sulfate with copper sulfate are obtained. This complex salt shows a potency of 630 mcg./mg. and [a] =+l02 (C. 0.48, H O). It further shows the antibacterial and antifungal activities.

EXAMPLE 3 When 0.63 gram (0.0025 mol.) of copper sulfate (CuSO -5H O) are added and dissolved into an aqueous solution of the kanamycin sulfate which was prepared in the same manner as in Example 1, the solution becomes deeply blue-violet colored. This solution is then treated as described in Example 1 and 3.5 grams of the complex salt of the kanamycin salt with copper sulfate are obtained as a violet-colored powdered.

This complex salt shows a potency of 698 meg/mg. and [a] =+l05 (C. 0.5, H 0).

EXAMPLE 4 When a solution of 2.5 grams (0.01 mol.) of copper sulfate (CuSO -5H O) dissolved in 7.5 cc. of distilled water is added to an aqueous solution of the kanamycin sulfate which was prepared in the same manner as in Example l, the aqueous solution becomes greenish-blue colored. This solution is allowed to leave overnight at the room temperature, filtered and then treated as described in Example 1. 3.3 grams of the complex salt of the kanamycin sulfate with copper sulfate are thus obtained as a greenish blue powder.

This complex salt shows a potency of 509 meg/mg. and [u] =+66.5 (C. 0.5, H 0).

(NiSO -7H O) in 5 cc. of water is added to 25 ml. of a waste lmother liquor which is obtained by removing the crystals of kanamycin sulfate from a filtrate of a kanamycin-fermentation beer and which contains about 0.0078 mol. of kanamycin B. The resulting solution shows a pH of 6.3 and the color of the solution changes from deep yellowish brown to deep yellowish green. Methanol is added to the solution and the precipitate formed is removed by filtration to yield 3.5 grams of a powder of greenish gray color. This powder shows the anti-bacterial, antifungal and anti-yeast activities.

The thus obtained complex salt comprising the kanamycin sulfate and nickel sulfate is expressed with the general formula:

C H N O -NiSO H SO -xH O where x is from 0 to 2, and shows the following analysis:

The minimum inhibition concentrations of this complex salt against various bacteria, fungi and yeasts are tabulated in Table 1 below.

Table 1 Kanamyein sulfate-nickel Medium Kanamyein A, sulfate com- 24 hrs, 'y/ml. plex salt, 24

hrs. -y/ml.

Acrobactcr acmgenes IAM 1102. Heart infusion 0.63 0. 63 Bacillus ccreus IAM 1110 1. 56 1.50 Escherichia coli LAM 1253 3. 13 8. 13 Micrococcus pygencs var aurcus 209 0. 31 0. 31 Proteus uulguris, 12. 5 0. Salmonella paralyp 0. 39 0. Salmonella paratg phi B 3. l3 3. 13 Streptococcus pygenes N 12. 5 12. 5 Mycobacterim 607 1 1. 56 1. 56 Saccharomycex cereuisiae s 1, 000 500 Saccharomyces rourli IFO 0845 250 500 Torula utilis 62. 5 62. 5 Willz'a 1 1 1, 000 500 Aspergillus m'ger 1 1, 000 500 Chetomium globosum ATCC 6205 1 250 125 Mom'lia silophila IFO 6070 250 250 Mucor pusz'llus IAM Mu14 250 500 Penlcillium chrysoganum Q 176 1, 000 1,000

1 Estimation is made after 48 hrs.

Moreover,the complex salt comprising the kanamycin sulfate and copper sulfate produced as described in the above is expressed with the general formula:

The minimum inhibition concentrations of this complex salt against various bacteria, fungi and yeasts are tabulated in Table 2 below.

Table 2-Continued 24 hrs. 'y/ml. 5 Salmonella paratyplzi B 1.56 Streptococcus pyogenes N.Y. 5 IID 12.5

Mycobacterium 607 3.13

where x is from 0 to 2, and shows the following analysis: Kanthomonae oryzae 0.5

. Saccharomyces rouxii IFO 0845 1 500.0

Calculated 30 Willia anomala 1 l000 Found Aspergillus n'iger 1 1000 i= Chetomium globosum ATCC 6205 1 1000 I Monilia sitophils IFO 6070 1 250.0

----------------------------------- 2 g 2% H2 Mucor pusillus IAM Mu-1-4 1 500.0

N 6139 7137 7120 Penicillium chrysogenum 0176 1 1000 36 Plzizopus nigricans IAM 37-39 1 500.0

1 Estimation is made after 48 hrs.

2 Estimation is made after 5 hrs.

capable of forming the complex salt with kanamycin, in

Table 2 addition to the nickel sulfate, copper chloride, there may 24 -1 'Y be also used nickel chloride, cobalt chloride, zinc chloride, Aerobacer aemgenes IAM 1102 Q78 magnesium sulfate, cadmium chloride, iron chloride, etc. Bacillus Cereus 1AM 1110 The activities of the complex salts of kanarnycin with Escherichia IAM 1253 313 these water-soluble metal salts also can be seen from the Micrococcus pyogenes var. aureus 209P 0,39 following Table 3 which shows the diameter of the in- Pmteus vulgaris 7,8 hibition zone of growth of various bacteria by means of Salmonella paratyphi A 0.78 paper-disc assay method.

Table 3 Bacillus Micrucoccus Pseudomonas subtilis ATCC pyoaencs var. aeruginosa, Mycobacte- 6633, mm. aureua 209P, mm. riuin 007, mm.

Kanamycin 21.2 20. 7 10. 5 18.6 21.8 20. 5 19. 0 18. 6 21. 0 21. 5 1s. 1 10.1 21.1 10.1 19.4

KanamyeimCuC]; complex salt 22. 0 21. 7 22. 0 21. 7 %.0 22. o 21. 0 22. 5 22. 0 22. 0 20. 3 21. 7 22.0 21.7 21.3

Kanamyeiu-NlCh complex salt 23. 0 21.0 21.0 20.0

Kanamycin-CoOl; complex salt 21. 7 21. 5 22. 0 22.0 20.3 20.7 22.0 19.7 20.3

Kanamycin-CdCh complex salt 22. 5 21. 7 21. 7 19. 8 22. 5 22. 0 22. 8 19.8 21.3 21 7 21.0 21.8 19.0

Norm-The complex salts are used as 0.2% solution.

What we claim is:

1. An antibacterial and antifungal composition for agricultural and fisheries applications, which contains as the active ingredient a complex salt of kanamycin and watersoluble inorganic metal salt containing a metal selected from the group consisting of copper, nickel, cobalt, zinc, cadmium, magnesium and iron, and an anion selected from the group consisting of chloride and sulfate and an aqueous carrier.

2. The process of inhibiting growth of bacteria and fungi in plant and marine life comprising applying to said plant and marine life a complex salt of kanamycin and Water-soluble inorganic metal salt containing a metal selected from the group consisting of copper, nickel, cobalt, zinc, cadmium, magnesium and iron, and an anion selected from the group consisting of chloride and sulfate and an aqueous carrier.

References Cited by the Examiner UNITED STATES PATENTS 2,777,791 1/1957 Visor et al. 167-16 2,793,974 5/1957 Rosen et al 167-30 2,908,606 10/1959 Gordon 167-33 2,931,798 4/1960 Utmezawa et a1 260-210 2,936,307 5/1960 Johnson et a1. 260-210 2,967,177 1/1961 Johnson et a1. 260-210 3,032,547 1/1962 Rothrock et al 260-210 LEWIS GOTTS, Primary Examiner.

MORRIS O. WOLK, Examiner. 

1. AN ANTIBACTERIAL AND ANTIFUNGAL COMPOSITION FOR AGRICULTURAL AND FISHERIES APPLICATIONS, WHICH CONTAINS AS THE ACTIVE INGREDIENT A COMPLEX SALT OF KANAMYCIN AND WATERSOLUBLE INORGANIC METAL SALT CONTAINING A METAL SELECTED FROM THE GROUP CONSISTING OF COPPER, NICKEL, COBALT, ZINC, CADMIUM, MAGNESIUM AND IRON, AND AN ANION SELECTED FROM THE GROUP CONSISTING OF CHLORIDE AND SULFATE AND AN AQUEOUS CARRIER. 